Cholesterol-dependent Syntaxin-4 and SNAP-23 Clustering Regulates Caveolar Fusion with the Endothelial Plasma Membrane

We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusi...

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Published in:The Journal of biological chemistry 2005-11, Vol.280 (44), p.37130-37138
Main Authors: Predescu, Sanda A., Predescu, Dan N., Shimizu, Kayo, Klein, Irene K., Malik, Asrar B.
Format: Article
Language:English
Subjects:
Biotin - metabolism
Caveolae - metabolism
Caveolin 1 - antagonists & inhibitors
Caveolin 1 - genetics
Caveolin 1 - metabolism
Cell Membrane - metabolism
Cell-Free System
Cholesterol - metabolism
Endothelium, Vascular - metabolism
Humans
Lung - metabolism
Membrane Fusion
Microscopy, Fluorescence
Qa-SNARE Proteins - metabolism
Qb-SNARE Proteins - metabolism
Qc-SNARE Proteins - metabolism
SNARE Proteins - metabolism
Streptavidin - metabolism
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Summary:We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M505659200